Herpes simplex virus type 1 (HSV-1) is a widespread human alpha herpesviral pathogen that causes a number of serious diseases including encephalitis, ocular keratitis, and disseminated neonatal infections. The ability of HSV-1 to maintain itself at high levels in the human population and to cause disease depends on viral spread, i.e., the transfer of infectious progeny virions to new cells and hosts. This occurs via two primary mechanisms: cell-cell spread and cell-free spread. HSV-1 cell-cell spread has been relatively well-studied, and several viral factors involved in this process are known. In contrast, cell-free spread has been little studied and there is scant knowledge of the mechanisms or viral factors involved. However, cell-free spread is critical to HSV-1 biology because it is almost certainly the mode of human-human transmission. Our recent published and unpublished data provide important clues to the mechanism of cell-free spread because they identify the first two HSV-1 factors specifically promoting cell-free virion release: the envelope glycoprotein C and the immediate-early protein ICP27. The data suggest that gC acts directly to enhance release, whereas ICP27 promotes gC expression, thereby indirectly enhancing release. In this grant application, we will investigate how these two viral proteins and possibly additional viral factors promote cell-free spread. Our underlying hypothesis is that the release of HSV-1 cell-free virions is directly driven by gC, and regulated by ICP27. To test our hypothesis, two Specific Aims are proposed. In Specific Aim 1, we will determine how gC promotes HSV-1 cell-free virion release. Two models will be tested that are consistent with the preliminary data. The first is that gC's heparan sulfate (HS)-binding activity allows progeny virions to bind to the cell surface and gain access to the cell-free release pathway. The second is that gC is required for the infectivity of cell-free virions, rather than for their release per se. In Specific Aim 2, we will elucidate how ICP27 regulates cell-free virion release. The model, based on preliminary data, is that ICP27 controls release by modulating the levels of gC that are incorporated into the envelope of progeny virions. It is proposed that ICP27 does this by modulating the removal of a small intron in the gC transcript, thus determining whether gC is produced as its full-length enveloped-associated form or as a secreted variant. The end result of this research will be increased knowledge about the mechanism and regulation of HSV-1 cell-free virion release. This knowledge will be relevant to the design of novel antivirals and therapies to mitigate HSV-1 disease and transmission. Moreover, increased understanding of cell-free release is likely to improve the efficacy and safety of HSV-1-based treatments, such as oncolytic HSV-1 strains that are currently being used clinically to treat cancer or viral vectors that are being developed for human gene therapy.